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Part:BBa_M50101:Experience

Designed by: Daniel Tang   Group: Stanford BIOE44 - S11   (2017-10-19)
Revision as of 23:35, 13 December 2017 by Danieldt (Talk | contribs)


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Applications of BBa_M50101

We used the optogenetic transcription factor EL-222 to drive transcription of another construct. We attached a p2A DasherGFP at the end of the other construct to infer transcription/expression activity.

Cells were transfected with these DNA constructs using Lipofectamine 3000 Reagent, Opti-MEM media, and P3000 (Thermo Fisher). Dual transfections with a maximum of 2μg of total DNA were added to each well at varying ratios of EL222 to C120-Insulin. Controls included single single transfections of EL222, C120-Insulin, GFP positive control (pHela), and a mock transfection. 24 hours post-transfection, fresh DMEM media was added. Effective transfection of pVP-EL222 was confirmed via microscopy. Light stimulation was then initiated for 24 hrs (465 nm, 8W/m^2,20s on-60s off continuous loop). Cells were kept at 37℃ and 5% CO2 during illumination.

We observed strong RFP expression in any cells with EL222 plasmid after only one day post-transfection using 1.5 ug of EL222 in our transfection.

RFPmic1.png

RFPmic2.png

From top to bottom: dual transfection (top), c120 only transfection (middle), el222 only (bottom)

RFP expression was retained two days post-transfection, as shown by flow-cytometry data. RFP was observed in dual transfection (blue and purple) and EL-222 only (red) cells but not in c120 only (green) or mock (grey) transfection groups.

RFPdual.png RFPexp.png

Y-axis: Normalized Count X-axis: RFP fluorescence

Using lower amounts (> 1 ug) of EL222 in transfection may lead to very low transfection efficiency, although more rigorous testing is required to prove this claim.

RFPlow.png

Grey - Mock, Red - No Light, Purple - 24 hr light (20s on, 60s off)

After 24 hrs of lighting, we were able to observe low efficiency GFP expression in dual transfected groups, but no in any of the controls.


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